Replica amplification of nucleic acid arrays

Abstract

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

What is claimed is:  
     
       1. A method of amplifying a plurality of nucleic acids, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers, wherein the primers are immobilized on a semi-solid support;  
       b) hybridizing amplification template to immobilized oligonucleotide primers,  
       c) extending the immobilized oligonucleotide primers using a DNA polymerase activity, and deoxynucleotide triphosphates to form immobilized nucleic acid strands and then denaturing the amplification template from the immobilized nucleic acid strands;  
       d) hybridizing non-immobilized oligonucleotide primers to the immobilized nucleic acid strands and extending :the non-immobilized oligonucleotide primers and denaturing the extended nonimmobilized oligonucleotide primers from the immobilized nucleic acid strands, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules.  
     
     
       2. A method of amplifying a plurality of nucleic acids, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers,  
       b) hybridizing amplification template to immobilized oligonucleotide primers,  
       c) extending the immobilized oligonucleotide primers using a DNA polymerase activity, and deoxynucleotide triphosphates to form immobilized nucleic acid strands and then denaturing the amplification template from the immobilized nucleic acid strands;  
       d) hybridizing non-immobilized oligonucleotide primers to the immobilized nucleic acid strands and extending the non-immobilized oligonucleotide primers and denaturing the extended nonimmobilized oligonucleotide primers from the immobilized nucleic acid strands, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules, wherein said non-immobilized oligonucleotide primer comprises a pool of oligonucleotide primers comprised of 5′ and 3′ sequence elements, said 5′ sequence element identical in all members of said pool, and said 3′ sequence element containing random sequences.  
     
     
       3. The method of  claim 2  wherein said 5′ sequence element comprises a restriction endonuclease recognition sequence. 
     
     
       4. The method of either of claims  2  or  3  wherein said 5′ element comprises a transcriptional promoter sequence. 
     
     
       5. A method of amplifying a plurality of nucleic acids, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers,  
       b) hybridizing amplification template to immobilized oligonucleotide primers,  
       c) extending the immobilized oligonucleotide primers using a DNA polymerase activity, and deoxynucleotide triphosphates to form immobilized nucleic acid strands and then denaturing the amplification template from the immobilized nucleic acid strands;  
       d) hybridizing non-immobilized oligonucleotide primers to the immobilized nucleic acid strands and extending the non-immobilized oligonucleotide primers and denaturing the extended nonimmobilized oligonucleotide primers from the immobilized nucleic acid strands, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules, wherein said immobilized oligonucleotide primers of said array of step (a) are heterogenous in nucleic acid sequence.  
     
     
       6. A method of amplifying a plurality of nucleic acids, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers,  
       b) hybridizing amplification template to immobilized oligonucleotide primers,  
       c) extending the immobilized oligonucleotide primers using a DNA polymerase activity, and deoxynucleotide triphosphates to form immobilized nucleic acid strands and then denaturing the amplification template from the immobilized nucleic acid strands;  
       d) hybridizing non-immobilized oligonucleotide primers to the immobilized nucleic acid strands and extending the non-immobilized oligonucleotide primers and denaturing the extended nonimmobilized oligonucleotide primers from the immobilized nucleic acid strands, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules, wherein said immobilized oligonucleotide primers are generated from genomic DNA.  
     
     
       7. A method of amplifying a plurality of nucleic acids, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers,  
       b) hybridizing amplification template to immobilized oligonucleotide primers,  
       c) extending the immobilized oligonucleotide primers using a DNA polymerase activity, and deoxynucleotide triphosphates to form immobilized nucleic acid strands and then denaturing the amplification template from the immobilized nucleic acid strands;  
       d) hybridizing non-immobilized oligonucleotide primers to the immobilized nucleic acid strands and extending the non-immobilized oligonucleotide primers and denaturing the extended nonimmobilized oligonucleotide primers from the immobilized nucleic acid strands, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules, wherein the array, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel.  
     
     
       8. The method of  claim 4  wherein the array, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel. 
     
     
       9. The method of  claim 5  wherein the array, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel. 
     
     
       10. The method of  claim 6  wherein the array, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel. 
     
     
       11. The method of either of claims  1  or  2  wherein said immobilized oligonucleotide primers of said array of step (a) are substantially pure in nucleic acid sequence. 
     
     
       12. The method of  claim 2  wherein said immobilized oligonucleotide primers of the array of step (a) are heterogenous in nucleic acid sequence. 
     
     
       13. The method of  claim 2  wherein said immobilized oligonucleotide primers are generated from genomic DNA. 
     
     
       14. The method of  claim 3  wherein said immobilized oligonucleotide primers are generated from genomic DNA. 
     
     
       15. The method of  claim 2  wherein the array, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel. 
     
     
       16. The method of  claim 3  wherein the immobilized oligonucleotide primers are generated from genomic DNA. 
     
     
       17. The method of  claim 1  wherein the non-immobilized oligonucleotide primer comprises a pool of oligonucleotide primers comprised of 5′ and 3′ sequence elements, said 5′ sequence element identical in all members of said pool, and said 3′ sequence element containing random sequences. 
     
     
       18. The method of  claim 17  wherein the 5′ sequence element comprises a restriction endonuclease recognition sequence. 
     
     
       19. The method of claims  17  wherein the 5′ element comprises a transcriptional promoter sequence. 
     
     
       20. The method of  claim 1  wherein the immobilized oligonucleotide primers of the array of step (a) are heterogenous in nucleic acid sequence. 
     
     
       21. The method of  claim 1  wherein the immobilized oligonucleotide primers are generated from genomic DNA. 
     
     
       22. The method of  claim 1  wherein the array, template, non-immobilized primer, and polymerase are cast in, a polyacrylamide gel. 
     
     
       23. The method  claim 1  wherein the immobilized oligonucleotide primers of the array of step (a) are substantially pure in nucleic acid sequence. 
     
     
       24. The method of  claim 1  wherein the non-immobilized oligonucleotide primers are heterogenous in nucleic acid sequence. 
     
     
       25. The method of  claim 1  wherein the non-immobilized oligonucleotide primers are substantially pure in nucleic acid sequence. 
     
     
       26. The method of  claim 1  wherein the immobilized oligonucleotide primers are heterogeneous with the non-immobilized oligonucleotide primers. 
     
     
       27. The method of  claim 1  wherein the immobilized oligonucleotide primers are substantially pure in nucleic acid sequence with the non-immobilized oligonucleotide primers. 
     
     
       28. A method of amplifying a nucleic acid molecule, said method comprising the steps of: 
       a) creating an array of immobilized oligonucleotide primers, wherein the primers are immobilized on a semi-solid support;  
       b) hybridizing amplification template to an immobilized oligonucleotide primer,  
       c) extending the immobilized oligonucleotide primer using a DNA polymerase activity, and deoxynucleotide triphosphates to form an immobilized nucleic acid strand and then denaturing the amplification template from the immobilized nucleic acid strand;  
       d) hybridizing a non-immobilized oligonucleotide primer to the immobilized nucleic acid strand and extending the non-immobilized oligonucleotide primer and denaturing the extended nonimmobilized oligonucleotide primer from the immobilized nucleic acid strand, and  
       e) repeating steps (b) and (c) and (d) for a defined number of cycles to yield a plurality of amplified nucleic acid molecules.